Much of modem biological research is concerned with identifying proteins involved in cellular processes and determining how, when, and where they are involved in specific biochemical pathways. However, despite recent advances in genome projects, the function of the majority of newly discovered genes remains unknown. There is now the pressing need to determine the functions of these novel gene products, such as those involved in disease phenotypes in humans or contributing to important agricultural traits in crop plants. The sequencing of the first genome of a higher plant, Arabidopsis, is now completed and sequencing programs for other plant genomes, such as rice, are in progress. These genomics programs will generate a wealth of information that is likely to offer new insights into important processes in plants and lead to exciting biotechnological applications in agriculture. However, it is in addressing questions of function where genomics-based research in plants and other organisms becomes bogged down and there is now the need for advances in the development of simple and automatable functional assays. Although many proteins have been identified by functional cloning of novel genes, such ‘expression cloning’ remains a significant experimental challenge. Many ingenious strategies have been devised to simultaneously screen cDNA libraries in the context of assays that allow both selection of clones and validation of their biological relevance.1-4 However, in the absence of an obvious functional assay that can be combined with cDNA library screening, researchers have turned to strategies that use as readout some general functional properties of proteins. A first step in defining the function of a novel gene is to determine its interactions with other gene products in an appropriate context; that is, since proteins make specific interactions with other proteins as part of functional assemblies, an appropriate way to examine the function of the product of a novel gene is to determine its physical relationships with the products of other genes. This is the basis, in part, of the highly successful Yeast Two-Hybrid system.5, 6 The success of this strategy in identifying biologically significant protein-protein interactions has been well documented, whether between two specific partners or between a “bait” and “prey” library.7, 8 
The central problem with two-hybrid screening is that detection of protein-protein interactions occurs in a fixed context, the nucleus of S. cerevisiae, and the results of a screening must therefore be further validated as biologically relevant using other assays in appropriate cell, tissue or organism models. While this would be true for any screening strategy, it would be advantageous if one could combine cDNA library screening with tests for biological relevance into a single strategy, thus eliminating false-positive interactions immediately. It was with this goal that a general strategy for detecting protein-protein interactions in intact cells based on Protein fragment Complementation Assays (PCA) was developed (11, 13, 14). In this strategy, the gene for an enzyme is rationally dissected into two pieces. Fusion proteins are constructed with two proteins that are thought to bind to each other, fused to either of the two probe fragments. Folding of the probe protein from its fragments is catalyzed by the binding of the test proteins to each other, and is detected as reconstitution of enzyme activity. The most advanced of these PCAs is one based on murine dihydrofolate reductase (mDHFR) (see FIG. 1 and discussion below).
There are several special features of the PCA strategy that makes it an interesting alternative to the Yeast Two-Hybrid approach: 1) PCA is “complete”; no other cellular activity is necessary and as a result a PCA can be done in any prokaryotic or eukaryotic cell type, or the PCA can be directed to a specific cellular compartment, organelle or membrane surface with the inclusion of appropriate signal sequences. 2) The portability of PCAs also means that induced versus constitutive protein-protein interactions can be distinguished by doing the PCA in a cell type where specific protein-protein interactions are thought to be induced by, for example a specific signal transduction pathway. 3) PCAs are not a single assay but a series of assays. The PCA strategy therefore has the added flexibility that an assay can be chosen because it works in a specific cell type appropriate for studying interactions of some class of proteins. 4) PCAs are inexpensive, requiring no specialized reagents beyond those necessary for a particular assay and off the shelf materials and technology. 5) PCAs can be automated and high-throughput screening could be done with little human intervention. 6) PCAs are designed at the level of the atomic structure of the enzymes used; because of this, there is additional flexibility in designing the probe fragments to control the sensitivity and stringencies of the assays. 7) PCAs can be based on enzymes for which the detection of protein-protein interactions can be determined differently.9 The simplest and most general approach is based on dominant selection, in which the reconstituted enzyme complements some missing metabolic enzyme in cells grown under selective pressure. Enzymes can also be chosen that produce a fluorescent or colored product for a more direct detection of protein-protein interactions. We have already developed 5 PCAs based on dominant-selection, colorimetric, or fluorescent outputs. Here we discuss the most well developed PCA, based on the enzyme murine dihydrofolate reductase (mDHFR) and its application to plants.10-14 We present results demonstrating the applicability of the DHFR to study molecular interactions in plant cells, allowing the detection of constitutive and induced protein-protein interactions, different methodologies that will be applicable to broad applications in agriculture and the screening of cDNA libraries for protein-protein interactions.
The DHFR PCA was the first we developed and is the most advanced in refinement and application12. The instant application describes in some detail the design principles and experimental strategy of the DHFR PCA as a selection strategy in E. coli, with particular emphasis on necessary controls to assure that the PCA detects protein-protein interactions and not some non-specific response of living cells to expression of the enzyme fragments. A number of mutants are studied as well as detailed kinetic studies of one of the reconstituted mutant enzymes. It also describes three specific examples of protein assembly that illustrate general uses of the assay strategy. The simplest example presented is detection of GCN4 leucine zipper forming sequences, followed by the more complex interaction of the p21 ras oncogene GTPase with its downstream signaling partner, the serine/threonine kinase raf. Finally, we demonstrate a natural product-mediated protein-protein interaction, that of the ternary complex of FKBP-rapamycin with the target of rapamycin FRB.
Applicants' have had considerable success in demonstrating the use of the DHFR PCA to rapidly screen and select for optimal leucine zipper-forming sequences in a two dimensional library by library screen; the first such example in the literature.10 These studies illustrate how the DHFR survival assay in E. coli is used to screen two libraries of complementary designed leucine zipper forming sequences each containing 105 clones, resulting in 1010 potential interacting pairs of which we could practically cover 106. The implications of these results are that not only does the selection strategy rapidly select for optimal properties of interacting sequences along with critical stereo- and regiospecific requirements for such complexes, but also for optimal in vivo characteristics, such as solubility and stability to proteolysis. The simplicity of this approach and specific nature of the information obtained about the design strategy suggest broad utility of the DHFR PCA in protein design and directed evolution experiments. It also shows that PCA rivals “phage display” strategies, since the entire selection, optimization and stringency tests are done in vivo, making this approach easily executed in almost any laboratory context. Most interesting are that given the sizes of the artificial libraries that we screened, by comparison, cDNA library screening with significant coverage would be feasible (for example, for libraries containing 103 to 105 unique cDNAs).
Recently, applicants have successfully demonstrated two different types of DHFR PCAs in mammalian cells13,14 and fortuitously, were able to apply them to a fundamental problem in growth factor membrane receptor biology.13 In one of these assays, the ‘DHFR PCA Survival Assay’, CHO DUKX-B11 (DHFR−) cells were co-transfected with DHFR complementary fragments F[1,2] or F[3] (FIG. 1, left) fused to two partner proteins. Co-transfectants were selected for survival in nucleotide-free medium (selection for DHFR activity). The assay has been demonstrated with GCN4 leucine zippers, the ras-raf complex, FKBP-rapamycin-FRB and the Erythropoietin (Epo) receptor and Epo Receptor-JAK2 kinase complexes.
In the second assay, the ‘DHFR fluorescence PCA’, the high-affinity fluorescein-conjugated DHFR inhibitor methotrexate (fMTX) passively diffuses into cells where it binds in a 1:1 complex with DHFR. Free fluorecein-methotrexate is actively transported from the cells leaving only DHFR-bound fMTX. In the DHFR PCA (FIG. 1, right), two proteins are fused to one of the two complementary fragments of DHFR (F[1,2] or F[3]) and coexpressed in a cell. If the two proteins interact, the DHFR fragments are brought into proximity and can fold/reassemble, rendering them capable of binding to fMTX. fMTX is retained in the cells and can be detected by fluorescence microscopy or fluorescence-activated flow cytometry. The first test system for the mammalian DHFR PCA was the pharmacologically well characterized rapamycin-induced association of FK506 binding protein (FKBP) to its target the FKBP-rapamycin binding domain of FRAP (FRB).16 The DHFR-negative CHO DUKX-B11 cells were stably co-transfected with FRB and FKBP fused to one of the two DHFR complementary fragments (FRB-F[1,2] and FKBP-F[3]). Co-transfectants were selected for survival in nucleotide-free medium (selection for DHFR activity) and in the presence of rapamycin. Only cells grown in the presence of rapamycin underwent normal cell division and colony formation (FIG. 2A). Survival was dependent only on the number of molecules of DHFR reassembled, and we determined that this number is approximately 25 molecules of DHFR per cell.14 
Formation of the FKBP-rapamycin-FRB complex was also detected in stably and transiently transfected cells with the fluorescence assay described above, based on stoichiometric binding of fluorescein-methotrexate to reconstituted DHFR in vivo. Fluorescence microscopy of unfixed co-transfected cells that had been incubated with fMTX showed high levels of fluorescence when cells were treated with rapamycin at saturating concentrations14. The fluorescence response of cell populations was quantified by FACS (FIG. 2B). The rapamycin-induced formation of FKBP/FRB was monitored by the shift in mean cell population fluorescence compared to non-induced cells. Quantitative rapamycin dose-dependence of this complex was demonstrated to be consistent with the known pharmacological response (FIGS. 2C, D). We have also used this approach to test a hypothesis for cytokine receptor activation suggested by the recent determination of the structure of native, unligated Epo receptor in the lab of Ian Wilson.13, 15 